A. Bioreactor Device
Animal cells and genetically altered derivatives thereof are often cultivated in bioreactors for the continuous production of vaccines, monoclonal antibodies, and pharmaceutical proteins such as hormones, antigens, tissue type plasminogen activators, and the like. For example, pituitary cells can be cultured in vitro to produce growth hormones; kidney cells can be cultured to produce plasminogen activators; and cultured liver cells have been known to produce hepatitis A antigen. In these bioreactors, cells are essentially a system of catalysts and the medium supplies and removes the nutrients and growth inhibiting metabolites. To supply nutrients and remove metabolites, the medium in-the bioreactor is changed either intermittently or continuously by fluid flow. However, because of their relatively small size and small density difference when compared to the medium, cells inevitably are withdrawn when the medium is changed, resulting in a relatively low cell concentration within the bioreactor. As a result of this low cell concentration, the concentration of the desired cell product is low in the harvested medium.
An ideal animal cell bioreactor would include three features:
(1) cells would be retained in a viable state at high densities in the bioreactor apparatus as long as possible, with an almost infinite residence time; PA1 (2) high molecular weight compounds, including expensive growth factors and the desired cell products, would have a long but finite residence time within the bioreactor to allow for both efficient nutrient utilization by the growing cells and also the accumulation of cell products to a high concentration; and PA1 (3) low molecular weight compounds, including less expensive nutrients and inhibitory substances, should have a very short residence time within the bioreactor to reduce inhibition of cell growth, cell product formation, and other cellular metabolic activities.
Numerous procedures and devices for in vitro cell culture production of biomolecules have attempted to achieve these goals in the past. In relatively simple systems, the cells have been grown in tissue flasks and roller bottles in the presence of a suitable nutrient media. More complex systems have used capillary hollow fiber membranes as a surface support for the cells in conjunction with a means for supplying nutrient media to the cells.
For example, U.S. Pat. No. 4,537,860 to Tolbert describes a static cell culture maintenance system for maintaining animal cells in a substantially arrested state of proliferation with continuous secretion of cell product. The cells are retained within a reactor vessel chamber in a semi-rigid matrix having interstices for passage of fluid nutrient medium. Fresh nutrient medium is supplied by perfusion into the matrix through relatively low porosity tubes which are suspended in the reactor chamber and which substantially traverse the matrix. High porosity tubes are available to withdraw expended medium and cell product.
A membrane-type cell reactor is also shown in "Construction of a Large Scale Membrane Reactor System with Different Compartments for Cells, Medium and Product", Develop. Bid. Standard., Vol. 66, pages 221-226 (1987). In this membrane system, cells are immobilized in a wire matrix where different membranes separate the cells from the medium and the cells from the cell product. The membrane lying between the medium and the cells is an ultrafilter with a useful molecular weight cut-off preventing the particular cell product from crossing into the medium compartment. The other membrane is a microfiltration membrane which separates the cells from a cell product chamber. With this configuration it is possible to feed the cells continuously and harvest the collected cell product at a distinct time interval without removing cells.
While these reactor systems attempt to tackle the problems of maintaining a high cell concentration to consequently harvest a high level of cell product, there is much room for improvement. Accordingly, the bioreactor of the present invention provides an in vitro cell culture system which maintains a large number of cells for an almost infinite residence time with continuous or intermittent cell product secretion.
B. Bioartificial Liver
Most patients admitted to an intensive care unit in liver failure do not survive. (Shellman, R. G.; Fulkerson, W. J.; DeLong, E.; Piantadosi, C. A. "Prognosis of patients with cirrhosis and chronic liver disease admitted to the medical intensive care unit". Crit Care Med; 1988 July; 16(7): 671-8.) Mortality as high as 80-90% has been reported. (Rueff, B.; Benhamou, J. P. "Acute hepatic necrosis and fulminant hepatic failure". GUT; 1983; 14: 805-15.) In 1987, more than twenty-six thousand people died of liver failure. Most of these deaths were not alcohol related. (Blake, J. E. ; Compton, K. V.; Schmidt, W.; Orrego, H. "Accuracy of death certificates in the diagnosis of alcoholic liver cirrhosis". Alcoholism (NY); 1988 February; 12(1): 168-72.)
The patient in hepatic failure, unlike the patient in renal failure, cannot be specifically treated. Renal dialysis, which revolutionized the treatment of renal failure, does not presently have a hepatic equivalent. Currently, the only available treatment for refractory liver failure is hepatic transplantation. Many patients in hepatic failure do not qualify for transplantation due to concomitant infection, or other organ failure. Because of organ shortages and long waiting lists, even those who qualify for liver transplantation often die while awaiting an allograft. UCLA reported that one quarter of their transplant candidates died before a liver could be obtained. Organs suitable for transplant in the pediatric age group are even scarcer. (Busuttil, R. W.; Colonna J. 0 2d; Hiatt, J. R.; Brems, J. J.; el Khoury G.; Goldstein, L. I.; Quinones-Baldrich, W. J.; Abdul-Rasool, I. H.; Ramming, K. P. "The first 100 liver transplants at UCLA". Ann Surg; 1987 October; 206(4): 387-402.)
Multiple Organ Failure Syndrome remains a major cause of death in the surgical intensive care unit. Hepatic failure is believed to be the dominant dysfunction. However, these patients die with histologically normal livers--except for cholestasis. Many investigators believe that outcomes could be improved with short-term hepatic support; the liver, and the patient, would recover given time.
Currently, other organ systems can be externally supported: left ventricular assist devices exist for the injured heart; dialysis units are used for kidney failure; parenteral nutrition is used for the nonfunctioning gastrointestinal tract; ventilators, extracorporeal membrane oxygenators, and veno-venous bypass techniques are employed to support lung function. However, there is currently no substitute for the liver, either to "buy time" for liver recovery or to find a suitable organ for transplantation.
The development of an artificial liver is a complex problem. Many prior attempts, such as plasmapheresis, charcoal and resin hemoperfusion, and xenograft cross circulation, have failed. Unlike the heart, that has one major physiological function, the liver performs many complex tasks necessary for survival. These tasks have been difficult to develop or maintain in mechanical systems.
The liver is the metabolic factory required for the biotransformation of both endogenous and exogenous waste molecules and the synthesis of glucose, lipids, and proteins--including albumin, enzymes, clotting factors, and carrier molecules for trace elements. The liver maintains appropriate plasma concentrations of amino and fatty acids, as well as detoxifying nitrogenous wastes, drugs, and other chemicals. Waste products, such as billrubin, are conjugated and excreted via the biliary tree. Hepatic protein synthesis and biotransformation vastly increase the complexity of hepatic support.
1. Culturing Hepatocytes
Systems that employ hepatocytes to provide biochemical function are problematic because hepatocytes can be difficult to maintain in culture. Under standard conditions, non-transformed hepatocytes cultured on plastic lose their gap junctions in about 12 to 24 hours; flatten, become agranular, and lose all their tissue specific functions in 3-5 days; and die within 1-2 weeks. (Reid, L. M.; Jefferson, D. M. "Culturing hepatocytes and other differentiated cells". Hepatology; 1984 May-June; 4(3): 548-59; Warren, M.; Fry, Jr. "Influence of medium composition on 7-alkoxycoumarin O-dealkylase activities of rat hepatocytes in primary maintenance culture". Zenobiotica; 1988 August; 18(8): 973-81).
A solution to this problem is the use of transformed hepatocytes because they can be grown much more easily. However, transformed hepatocytes are often considered a poor choice because even well-differentiated transformed cells show marked variations in tissue specific function from their parent tissues. (Reid, et al., 1984, supra.) Moreover, many cell lines are transformed by viruses. (Aden, D. P.; Fogel, A.; Plotkin, S.; Damjanov, I.; Knowles, B. B. "Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line". Nature, 1979 Dec. 6 :61 5-6; Knowles, B. B.; Howe, C. C.; Aden, D. P. "Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen". Science; 1980 July 25; 209: 497-9.) These cell lines have the potential to transmit the transforming virus to the patient. As a result, it is doubtful that regulatory agencies would approve the use of transformed cells for humans, even if the risk of transmission were proven minimal.
Many approaches to prolonging the viability and function of cultured hepatocytes and other differentiated cells have been investigated. These approaches have included adding hormones and growth factors to the culture media, adding extracellular matrix constituents, and growing the hepatocytes in the presence of another cell type. Cells routinely used in co-culture work with hepatocytes are endothelial cells, or hepatic nonparenchymal cells such as Kupffer cells.
2. Effect of Hormones and Growth Factors
The addition of corticosteroids to the incubation media has been shown to prolong survival of cultured hepatocytes and to maintain albumin synthesis--particularly in synergy with insulin. (Jefferson, D. M.; Clayton, D. F.; Darnell, J. E. Jr.; Reid, L. M. "Post-transcriptional modulation of gene expression in cultured rat hepatocytes". Mol Cell Biol; 1984 September; 4(9): 1929-34; Dich, J.; Vind, C.; Grunnet, N. "Long-term culture of hepatocytes: effect of hormones on enzyme activities and metabolic capacity". Hepatology; 1988 January-February; 8(1): 39-45.) DMSO (Dimethyl sulfoxide) and phenobarbital also are known to prolong hepatocyte viability and function. (Maher, J. J. "Primary hepatocyte culture: is it home away from home?" Hepatology; 1988 September-October; 8(5): 1162-6.) Not all tissue specific functions are equally supported, however. Insulin can promote some functions with an effect that varies with concentration. If only insulin is added to the medium, urea cycle enzyme expression is decreased. This negative effect can be counteracted by the addition of glucagon and dexamethasone. (Dich, et al., 1988, supra.)
Hormonally defined media can also prolong hepatocyte function and viability. (Jefferson, et al., 1984, supra.) Using a serum-free hormonally defined medium, good function in baboon hepatocytes has been shown for over 70 days. This medium consisted of epidermal growth factor (100 ng/ml), insulin (10 .mu.g/ml), glucagon (4 mg/ml), albumin (0.5 mg/ml), linoleic acid (5 mg/ml), hydrocortisone (10.sup.-6 M), selenium (10.sup.-7 M), cholera toxin (2 ng/ml), glycyl-histidyl-lysine (20 ng/ml), transferrin (5 mg/ml), ethanolamine (10.sup.-6 M), prolactin (100 ng/ml), somatotropin (1 mg/ml), and thyrotropin releasing factor (10.sup.-6 M). (Lanford, L. E.; Carey, K. D.; Estlack, L. E.; Smith, G. C.; Hay, R. V. "Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium". In Vitro Cell Dev Biol; February 1989; 25(2): 174-82.)
3. Effect of Matrices
It is now clear that the extracellular matrix has considerable influence on cell function and survival. (Bissell, M. J.; Aggeler, J. "Dynamic reciprocity: How do extracellular matrix and hormones direct gene expression". Mechanisms of Signal Transduction by Hormones and Growth Factors: Alan R. Liss, Inc.; 1987: 251-62.3.) Matrix elements have been shown to reduce or obviate the need for specific growth factors. Using extracted hepatic connective tissue, hepatocytes have been cultured for over 5 months and maintained albumin synthesis for at least 100 days. This extract represented approximately 1% of the liver by weight. One third of the extract was composed of carbohydrates and noncollagenous proteins; the other two thirds were collagens--43% Type I, 43% Type III, and the remainder an undefined mixture of others including Type IV. (Rojkind, M.; Gatmaitan, Z.; Mackensen, S.; Giambrone, M.; Ponce, P.; Reid, L. "Connective tissue Biomatrix: Its Isolation and Utilization for Long-term Cultures of Normal Rat Hepatocytes". J Cell Biol; October 1980; 87: 255-63.) This mixture may not accurately reflect the local hepatocyte environment--the peri-sinusoidal space or Space of Disse.
The presence of matrix in the Space of Disse has been controversial. Some researchers initially suggested that the peri-sinusoidal space was "empty." It is now appreciated that all of the major constituents of basement membrane are present in or around the Space of Disse. (Bissell, D. M.; Choun, M. O. "The role of extracellular matrix in normal liver". Scand. J. Gastroenterol.; 1988; 23(suppl 151): 1-7.)
Heparan sulfate proteoglycan binds both cell growth factors and cells. (Saksela, O.; Moscatelli, D.; Sommer, A.; Rifkin, D. B. "Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation". J Cell Biol; 1988 August; 107(2): 743-51; Gordon, M. Y.; Riley, G. P.; Clarke, D.; "Heparan sulfate is necessary for adhesive interactions between human early hemopoietic progenitor cells and the extracellular matrix of the marrow microenvironment". Leukemia 1988 December; 2(12): 804-9.) Heparan sulfate may directly effect the hepatocyte nucleus. (Ishihara, M.; Fedarko, N. S.; Conrad, H. E. "Transport of heparan sulfate into the nuclei of hepatocytes"; J Biol Chem; 1986 October 15; 261(29): 13575-80.) Hepatocytes secrete relatively abundant quantities of heparan sulfate in culture. (Arenson, D. M.; Friedman, S. L.; Bissell, D. M. "Formation of extracellular matrix in normal rat liver: lipocytes as a major source of proteoglycan". Gastroenterology; 1988 August; 95(2): 441-7.) Immunological studies have identified Type I collagen, Type III collagen, Type IV collagen, fibronectin, and laminin in the Space of Disse. (Geerts, A.; Geuze, H. J.; Slot, J. W.; Voss, B.; Schuppan, D.; Schellinck, P.; Wisse, E. "Immunogold localization of procollagen III, fibronectin and heparan sulfate proteoglycan on ultrathin frozen sections of the normal rat liver". Histochemistry; 1986; 84(4-6): 355-62; Martinez-Hernandez, A. "The hepatic extracellular matrix. I. Electron immunohistochemical studies in normal rat liver". Lab Invest;, 1984 July; 51(1): 57-74.) There is normally little Type I collagen in the Space of Disse, although hepatocytes in culture show increasing Type I synthesis with de-differentiation. This is at the expense of Type III collagen synthesis. This effect is reversed with culture techniques that support tissue specific hepatocyte activity.
Hepatocytes also can be cultured on MATRIGEL.TM., a biomatrix produced by a sarcoma cell line (EHS). MATRIGEL contains Type IV collagen, laminin, entactin, and heparan sulfate. On MATRIGEL, hepatocytes have been shown to maintain normal albumin synthesis for 21 days. (Bissell, et al., 1987, supra.)
Close duplication of the normal environment of the hepatocyte has also been attempted by culturing hepatocytes in a confluent monolayer on collagen. A second layer of Type I collagen is added to recreate the normal matrix "sandwich" formed on the "top" and on the "bottom" of the hepatocyte. This technique has shown significantly improved viability and function with albumin synthesis for more than 42 days. (Dunn, J. C. Y.; Yarmush, M. L.; Koebe, H. G.; Tompkins, R. G. "Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration". FASEB; 1989 February; 3: 174-7.)
The effect of various proteoglycans and glycosaminoglycans on gap junction protein synthesis and genetic expression has also been carefully examined. The most effective compounds were dermatin sulfate proteoglycan, chondroitin sulfate proteoglycan, and heparan. Heparan extracted from the liver was most effective. Lambda carrageenan, a seaweed extract, was also effective. (Spray, D.C.; Fujita, M.; Saez, J. C.; Choi, H.; Watanabe, T.; Hertzberg, E.; Rosenberg, L. C.; Reid, L. M. "Proteoglycans and Glycosaminoglycans Induce Gap Junction Synthesis and Function in Primary Liver Cultures". J Cell Biol; 1987 July; 105: 541-55.) Finally, chitosan, a polysaccharide found in crustacean shells and fungal membranes, has been suggested as a factor that can mimic normal matrix and promote function and survival. (Muzzarelli, R.; Baldassarre, V.; Conti, F.; Ferrara, P.; Biagini, G.; Gazzanelli, G.; Vasi, V. "Biological activity of chitosan: ultrastructural study". Biomaterials; 1988 May, 9(3): 247-52; Scholz, M. T.; Hu, W-S. "A two compartment cell entrapment bioreactor with three different holding times for cells, high and low molecular weight compounds". Cytotechnology 4: 127-137, 1990.)
4. Cell-Cell Co-Culture
Another successful technique for culturing differentiated liver cells involves co-culturing them with nonparenchymal cells. Recently, co-culture of hepatocytes on various endothelial lines was compared. Co-culture showed significantly improved albumin synthesis and maintenance of gap junctions. The cells were grown in the presence of insulin and dexamethasone. The addition of serum did not improve the results. The improved survival and function conferred by co-culture occurred only with cells in close proximity, and was not transferred by cell supernatants. (Goulet, F.; Normand, C.; Morin, O. "Cellular interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocytes". Hepatology; 1988 September-October; 8(5): 1010-8.)
It is still controversial whether the beneficial effects of co-culture occur through matrix interactions or require cell-cell contact.
There is also evidence that lipocytes play a key role in matrix production. Lipocytes are reported to be as numerous as Kupffer cells, and have been suggested to produce the majority of Type I collagen, Type II collagen, Type IV collagen, laminin, and proteoglycans--particularly dermatin sulfate proteoglycan and chondroitin sulfate proteoglycan. (Friedman, S. L.; Roll, F. J.; Boyles, J.; Bissell, D. M. "Hepatic lipocytes: The principle collagen-producing cells of normal rat liver". PNAS; December 1985; 82: 8681-5.) It is of particular interest that these specific proteoglycans were those that best support gap junctions (Spray, et al., 1987, supra.).
5. Bioartificial Liver--Previous Investigations
Many techniques of artificial support have been utilized over the past three and a half decades. These include simple exchange transfusions (Lee, C.; Tink, A. "Exchange transfusion in hepatic coma: report of a case". The Med. J. Australia; 1958, January 11: 40-42; Trey, C.; Burns, D. G.; Saunders, S. J. "Treatment of hepatic coma by exchange blood transfusion". NEJM; 1966; 274(9): 473-81); plasmapheresis with plasma exchange; (Sabin. S., Merritt, J. A. "Treatment of hepatic coma in cirrhosis by plasmapheresis and plasma infusion [plasma exchange]". Annals of Internal Medicine; 1968 January; 68(1): 1-6); extracorporeal heterologous or homologous liver perfusion (Elsemann, B.; Liem, D. S.; Raffucci, F. "Heterologous liver perfusion in treatment of hepatic failure". Annals of Surgery, 1965; 162(3): 329-345; Sen, P. K.; Bhalerao, R. A.; Parulkar, G. P.; Samsi, A. B., Shah, B. K.; Kinare, S. G. "Use of isolated perfused cadaveric liver in the management of hepatic failure". Surgery; 1966, May; 59(5): 774-781); cross-circulation (Burnell, J. M.; Dawlorn, J. K.; Epstein, R. B.; Gutman, R. A.; Leinbach, G. E.; Thomas, E. D.; Volwiler, W. "Acute hepatic coma treated by cross-circulation or exchange transfusions". NEJM; 1967; 276(17): 943-953); hemodialysis (Opolon, P.; Rapin, J. R.; Huguet, C.; Granger, A.; Delorme, M. L.; Boschat, M.; Sausse, A. "Hepatic failure coma (HFC) treated by polyacrylonitrile membrane (PAN) hemodialysis (HD)". Trans. ASAIO; 1976; 22: 701-710); activated charcoal hemoperfusion (Gazzard, B. G.; Weston, M. J.; Murray-Lyon, I. M.; Flax, H.; Record, C. O.; Portmann, B.; Langley, P. G.; Dunlop, E. H.; Mellon, P. J.; Ward, M. B.; Williams, R. "Charcoal haemoperfusion in the treatment of fulminant hepatic failure". Lancet, June 29; i: 1301-1307); and, more recently, bioartificial liver systems containing cultured hepatocytes.
Examples of bioartificial liver systems currently being investigated for support of liver failure include extracorporeal bioreactors (Arnaout, W. S.; Moscioni, A. D.; Barbour, R. L.; Demetriou, A. A. "Development of bioartificial liver: bilirubin conjugation in Gunn rats". Journal of Surgical Research; 1990; 48: 379-382; Margulis, M. S., Eruckhimov, E. A.; Ahdieimann, L. A.; Viksna, L. M. "Temporary organ substitution by hemoperfusion through suspense of active donor hepatocytes in a total complex of intensive therapy in patients with acute hepatic insufficiency". Resuscitation; 1989; 18: 85-94); and implantable hepatocyte cultures, such as microencapsulated gel droplets (Cai, Z.; Shi, Z.; O'Shea, G. M.; Sun, A. M. "Microencapsulated hepatocytes for bioartificial liver support". Artificial Organs; 1988 May; 12(5): 388-393) and spheroid aggregates (Saito, S.; Sakagami, K.; Koide, N.; Morisaki, F.; Takasu, S., Oiwa, T., Orita, K. "Transplantation of spheroidal aggregate cultured hepatocytes into rat spleen". Transplantation Proceedings; 1989 February; 21(1): 2374-77). These bioartificial liver systems have the advantage of performing detoxification, synthesis and bioprocessing functions of the normal liver. Only a few extracorporeal bioreactors have been used in the clinical setting (Matsumura, K. N.; Guevara, G. R.; Huston, H.; Hamilto, W. L.; Rikimaru, M.; Yamasaki, G.; Matsumura, M. S. "Hybrid bioartificial liver in hepatic failure: preliminary clinical report". Surgery;, 1987 January; 101(1): 99-103; Margulis, et al.; 1989, supra). Implantable hepatocyte cultures remain clinically untested.
The technique for hepatocyte entrapment within microencapsulated gel droplets (hepatocyte microencapsulation) is similar to the technique successfully used for pancreatic islet encapsulation (O'Shea, G. M.; Sun, A. M. "Encapsulation of rat islets of Langerhans prolongs xenograft survival in diabetic mice". Diabetes; 1986 August; 35: 943-46; Cai, et al., 1988, supra). Microencapsulation allows nutrient diffusion to the hepatocytes, and metabolite and synthetic production diffusion from the hepatocytes. Microencapsulation also provides intraperitoneal hepatocytes with "immuno-isolation" from the host defenses (Wong, H.; Chang, T. M. S. "The viability and regeneration of artificial cell microencapsulated rat hepatocyte xenograft transplants in mice". Biomat. Art. Cells, Art. Org.; 1988; 16(4): 731-739.) Plasma protein and albumin synthesis (Sun, A. M.; Cai, Z.; Shi, Z.; Fengzhu, M.; O'Shea, G. M.; Gharopetian, H. "Microencapsulated hepatocytes as a bioartificial liver". Trans. ASAIO; 1986; 32: 39-41; Cai, et al., 1988, supra); cytochrome P450 activity and conjugation activity (Tompkins, R. G.; Carter, E. A.; Carlson, J. D.; Yarmush, M. L. "Enzymatic function of alginate immobilized rate hepatocytes". Biotechnol. Bioeng.; 1988; 31: 11-18); gluconeogenesis (Miura, Y.; Akimoto, T.; Yagi, K. "Liver functions in hepatocytes entrapped within calcium alginate". Ann. N.Y. Acad. Sci.; 1988; 542: 531-32); ureagenesis (Sun, A. M.; Cai, Z.; Shi, Z.; Ma, F.; O'Shea, G. M. "Microencapsulated hepatocytes: an in vitro and in vivo study". Biomat. Art. Cells, Art. Org.; 1987; 15: 483-486); and hepatic stimulating substance production (Kashani, S. A.; Chang, T. M. S. "Release of hepatic stimulatory substance from cultures of free and microencapsulated hepatocytes: preliminary report". Biomat., Art Cells, Art. Org.; 1988; 16(4): 741-746) have all been reported by calcium alginate entrapped hepatocytes.
Spheroid aggregate cultured hepatocytes have also been proposed for the treatment of fulminant hepatic failure. Multiple techniques exist for hepatocyte aggregation into spheroids (Saito, S.; Sakagami, K.; Koide, N.; Morisaki, F.; Takasu, S.; Oiwa, T.; Orita, K. "Transplantation of spheroidal aggregate cultured hepatocytes into rat spleen". Transplantation Proceedings; 1989 February; 21(1): 2374-77; Koide, N.; Shinji, T.; Tanube, T.; Asano, K.; Kawaguchi, M.; Sakaguchi, K.; Koide, Y.; Mori, M.; Tsuji, T. "Continued high albumin production by multicellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans". Biochem. Biophys. Res. Comm.; 1989; 161(1): 385-91.) It is hypothesized that hepatocyte aggregation would improve the beneficial results of intraperitoneal hepatocyte injection therapy.
Extracorporeal bioreactor designs for the purpose of artificial liver support have included perfusion of small liver cubes (Lie, T. S., Jung, V., Kachel, F., Hohnke, C., Lee KS. "Successful treatment of hepatic coma by a new artificial liver device in the pig". Res. Exp. Med.; 1985; 185: 483-494); dialysis against a hepatocyte suspension (Matsumura, et al., 1987, supra; Margulis, et al., 1989, supra); perfusion of multiple parallel plates (Uchino, J.; Tsuburaya, T.; Kumagai, F.; Hase, T.; Hamoda, T.; Komai, T.; Funatsu, A.; Hashimura, E.; Nakamura, K.; Kon, T. "A hybrid bioartificial liver composed of multiplated hepatocyte monolayers". Trans. ASAIO; 1988; 34: 972-977); and hollow fiber perfusion. Human studies using extracorporeal hepatocyte suspensions have been reported.
The first clinical report of artificial liver support by dialysis against a hepatocyte suspension was released in 1987 (Matsumura, et al., 1987, supra). The device consisted of a rabbit hepatocyte liquid suspension (1-2 liters) separated from the patient's blood by a cellulose acetate dialysis membrane. Each treatment used fresh hepatocytes during a single four to six hour dialysis (run). Multiple runs successfully reduced serum bilirubin and reversed metabolic encephalopathy in a single case.
A controlled study from the USSR comparing dialysis against a hepatocyte suspension with standard medical therapy for support of acute liver failure was recently reported (Margulis, et al.; 1989, supra). The bioartificial device consisted of a small 20 ml cartridge filled with pig hepatocytes in liquid suspension, along with activated charcoal granules. The cartridge was perfused through a Scribner arteriovenous shunt access. Patients were treated daily for six hours. The hepatocyte suspension was changed hourly over each six hour treatment period. Improved survival was demonstrated in the treated group (63%) when compared with the standard medical therapy control group (41%).
Culturing hepatocytes with a hollow fiber cartridge is another example of bioartificial liver support. Traditionally, hepatocytes are loaded in the extracapillary space of the hollow fiber cartridge, while medium, blood or plasma is perfused through the lumen of the hollow fibers. Cells may be free in suspension (Wolf, C. F. W.; Munkelt, B. E. "Bilirubin conjugation by an artificial liver composed of cultured cells and synthetic capillaries". Trans. ASAIO; 1975; 21: 16-27); attached to walls (Hager, J. C.; Carman, R.; Stoller, R.; Panol, G.; Leduc, E. H.; Thayer, W. R.; Porter, L. E.; Galletti, P. M.; Calabresi, P. "A Prototype For A Hybrid Artificial Liver". Trans. ASAIO; 1978; 24: 250-253); or attached to microcarriers which significantly increase the surface area within the extracapillary space (Arnaout, et al., 1990, supra).
Bilirubin uptake, conjugation and excretion by Reuber hepatoma cells within a hollow fiber cartridge was reported in 1975. (Wolf, et al., 1975, supra). Tumor cell suspensions were injected by syringe into the shell side of the compartment while bilirubin containing medium was perfused through the hollow fiber intraluminal space. This technique has not been reported clinically, possibly due to the risk of tumor seeding by hepatoma cells.
Another hollow fiber device developed for liver support uses hepatocytes attached to microcarriers loaded into the extracapillary cavity of a hollow fiber cartridge. In this device, blood flows through semi-permeable hollow fibers allowing the exchange of small molecules. Using this system, increased conjugated bilirubin levels have been measured in the bile of glucuronosyl transferase deficient (Gunn) rats. (Arnaout, W. S.; Mosicioni, A. D.; Barbour, R. L.; Demetriou, A. A. "Development of Bioartificial Liver: Bilirubin Conjugation in Gunn Rats". J. Surg. Research; 1990; 48: 379-82.) Since the outer shell is not perfused, all oxygen and nutrients are provided by the patient's blood stream. In addition, this system may require an intact in vivo biliary tree for the excretion of biliary and toxic wastes.